Genome-Wide CRISPR Screen Reveals Autophagy Disruption as the Convergence Mechanism That Regulates the NRF2 Transcription Factor

ABSTRACT

The nuclear factor (erythroid 2)-like 2 (NRF2 or NFE2L2) transcription factor regulates the expression of many genes that are critical in maintaining cellular homeostasis. Its deregulation has been implicated in many diseases, including cancer and metabolic and neurodegenerative diseases. While several mechanisms by which NRF2 can be activated have gradually been identified over time, a more complete regulatory network of NRF2 is still lacking. Here we show through a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen that a total of 273 genes, when knocked out, will lead to sustained NRF2 activation. Pathway analysis revealed a significant overrepresentation of genes (18 of the 273 genes) involved in autophagy. Molecular validation of a subset of the enriched genes identified 8 high-confidence genes that negatively regulate NRF2 activity irrespective of cell type: ATG12, ATG7, GOSR1, IFT172, NRXN2, RAB6A, VPS37A, and the well-known negative regulator of NRF2, KEAP1. Of these, ATG12, ATG7, KEAP1, and VPS37A are known to be involved in autophagic processes. Our results present a comprehensive list of NRF2 negative regulators and reveal an intimate link between autophagy and NRF2 regulation.

KEYWORDS:

• CRISPR
• KEAP1
• NFE2L2
• NRF2
• autophagy
• gene reporters
• genomics
• molecular biology
• oxidative stress
• signal transduction

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00037-19.

ACKNOWLEDGMENTS

We declare that we have no conflicts of interest.

This work was supported by grant R21ES027920 (A.O.) from the National Institute of Environmental Health Sciences. A.O. was supported in part by R01CA226920. D.D.Z. was supported in part by R01DK109555, R01ES026845, and P42ES004940. Both D.D.Z. and A.O. are members of the P30ES006694-funded center. M.J.K. is supported by the National Science Foundation Graduate Research Fellowship Program under grant no. DGE-1746060.

Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

We thank the UCLA TCGB sequencing core under the direction of Xinmin Li for technical advice.